PROJECT SUMMARY/ ABSTRACT: Cardiac neural crest cells are crucial for the development of the cardiovascular system by contributing to a small proportion of cardiomyocytes of the adult heart. This unique population of neural crest-derived cardiomyocytes represents an appealing source of precursor-like cells able to control myocardial regeneration. My published and preliminary data show that upon injury sox10+ cells expand in an injury-induced proliferative manner and are required for successful regeneration by initiating a developing neural crest gene regulatory network. While preliminary results suggest it is likely these cells are derived from a neural crest origin, this has yet to be explored at great depth. Previous studies of cardiac regeneration have focused primarily on mesoderm- derived populations, and, now, much remains to be uncovered about the role of ectoderm-derived neural crest cells in the repair process. With this Pathway to Independence Award, I will seek to understand the underlying mechanisms of neural crest contributions to adult heart repair using cell biological approaches, systems-level techniques, and high-resolution imaging of regenerating hearts. The overall objectives of this proposal are to test the hypothesis that neural crest-derived cells are required for heart regeneration in zebrafish (Aim 1), use single cell RNA-seq and ChIP-seq to look for subpopulations of neural crest-derived cells that express distinct gene signatures in response to injury (Aim 2), and investigate, at high-resolution, the morphogenetic events that allow adult sox10+ cells to contribute to the injury response (Aim 3). The central hypothesis of this proposal is that neural crest-derived cells play an essential role in heart regeneration by redeploying a developmental neural crest program. The long term goal of this project is to understand how developmental programs are reused for regeneration in the adult body and how we can hijack these gene regulatory circuits for reprogramming cell types that do not regenerate in order to have a more regenerative potential. The work proposed in this Pathway to Independence Award proposal will be greatly facilitated by my multi-disciplinary advisory committee with expertise in tissue regeneration, single cell RNA-seq approaches and analyses, and genome biology. After developing a formidable skillset and research foundation afforded by the two years of the mentored phase of this award, my goal is to establish a high impact, independent research group that will combine systems-level approaches with state-of-the-art cell and developmental biology techniques to answer questions of regeneration through a developmental lens. Long term project hypotheses are focused on how we can regenerate the human heart. My preliminary data shows cardiac neural crest cells contribute to cardiomyocytes not only in fish but also in amniotes; therefore, the mechanisms that stimulate these cells to contribute to regeneration in zebrafish may offer therapeutic approaches to repair heart damage in mammals including humans.